Abstract Recent studies of the expression of the Staphylococcus aureus cid and lrg operons during biofilm development have demonstrated the existence of distinct metabolic microniches that are proposed to serve different biological roles. Unfortunately, a critical barrier to understanding these potential roles is the difficulty in separating the microniche cells from other regions of the biofilm so that they can be subjected to various analytical methods. Thus, the overall objective for this project is to apply cellular separation techniques to isolate the microniche cells and then subject them to comprehensive transcriptomic analyses to gain a better understanding of their basic characteristics. The central hypothesis that drives this research is that the architecture of the S. aureus biofilm is produced by changes in metabolism, thus forming distinct functional microniches. The rationale for this project is that understanding the metabolic and functional heterogeneity associated with biofilm growth will provide new insights into the biology of this pathogenic organism leading to improved therapeutic strategies. Guided by preliminary data, this objective will be achieved by attaining two specific aims: 1) Identify transcriptional changes that critically distinguish different microniches and 2) Examine the functions of differentially expressed genes in biofilm development. Under the first aim, microniches (micro-colonies and basal layer) will be identified by tracking expression of fluorescent cid and lrg reporter fusions and separated using the complementary techniques of flow cytometry and laser-capture microdissection microscopy. RNA will be extracted from the microniches and gene expression differentiated and quantified via RNAseq. In the second aim, genes found to be differentially expressed and genes regulated by the LytSR two-component system will be tested for their effects on micro-colony formation in a microfluidic system (BioFlux). This contribution will be significant because it is expected to have a significant impact on our understanding of S. aureus biofilm maturation. The proposed research is innovative because it represents a departure from the status quo, which averages gene expression from the entire biofilm, by providing a comprehensive transcriptomic microniche-specific analysis of the S. aureus biofilm and validation of genes identified as specific to a microenvironment using a microfluidic device (the Bioflux) and subsequent interrogation using the Nebraska Transposon Mutant Library. The expected outcome of the work described in this proposal is an enhanced understanding of the development of biofilms produced by S. aureus, and the metabolic diversity inherent to these structures.